CN108267594A - A kind of ST2 detection kits, method of preparation and use based on bimolecular fluorescence complementary technology - Google Patents
A kind of ST2 detection kits, method of preparation and use based on bimolecular fluorescence complementary technology Download PDFInfo
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- CN108267594A CN108267594A CN201711271870.2A CN201711271870A CN108267594A CN 108267594 A CN108267594 A CN 108267594A CN 201711271870 A CN201711271870 A CN 201711271870A CN 108267594 A CN108267594 A CN 108267594A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
- G01N33/6869—Interleukin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/52—Assays involving cytokines
- G01N2333/54—Interleukins [IL]
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Abstract
The present invention provides a kind of ST2 detection kits, method of preparation and use based on bimolecular fluorescence complementary technology.The kit includes：The fluorescin N-terminal segment of anti-ST2 antibody couplings, the fluorescin C-terminal segment of anti-ST2 antibody couplings；The invention also discloses a kind of preparation method of the ST2 diagnostic kits based on bimolecular fluorescence complementary technology, this method includes：The preparation of the fluorescin N-terminal segment of anti-ST2 antibody couplings, the preparation of the fluorescin C-terminal segment of anti-ST2 antibody couplings；Finally also disclose the application method of the kit；Kit of the present invention has many advantages, such as that easy to operate, the range of linearity is wide, specificity is good, free of cleaning, accuracy is high, it is used convenient for clinical detection, it is applied to the monitoring that the ability effect of Severity of Coronary Lesion occurs and assesses for prediction acute myocardial infarction AMI (AMI) patient's prognosis mala cardiovascular event, clinician is improved to the clinical evaluation of AMI patient or AMI people at highest risk and risk stratification accuracy rate, there is great market value.
Description
Technical field
The content of ST2 in human body is detected for ion vitro immunization diagnosis the present invention relates to a kind of bimolecular fluorescence complementary technology,
Belong to medical diagnosis on disease detection field.
Background technology
2 albumen of growth stimulation expressing gene (growth stimulation expressed gene 2, ST2) is IL-1
Receptor superfamily member was had found in 1989 by Tominaga, is considered as an orphan receptor and inflammation for a long time at first
Ligands specific IL-33 related to immunity disease, being found that it by 2005, then expands to one to the research of ST2
New field.The gene of people ST2 is located at chromosome 2q12, about 40KD, is expressed in mast cell, macrophage, the auxiliary activated
Property T cell 2 (Th2), cardiac muscle cell and Cardiac Fibroblasts.
ST2 genes have 4 kinds of transcription products, wherein 2 most important hypotypes are cross-module type ST2 (ST2L) and solubility ST2
(sST2), it is to be formed by Promoter selectivity shearing and 3 ' end processing.It is by 3 companies that ST2L, which includes an extracellular domain,
Continuous immunoglobulin die body composition, a transmembrane domain and Toll/IL-1 receptors (TIR) intracellular domain；With
ST2L is compared, and sST2 missing cross-films and intracellular domain are only made of, Ke Yifen a C-terminal sequence containing 9 amino acid
It secretes extracellular；And ST2V lacks the 3rd immunoglobulin die body, and forms one in C-terminal alternative splicing and special dredge
Pool；ST2LV is that alternative splicing has fallen the transmembrane domain of ST2L and formed.
ST2 genes when cardiac muscle cell and Cardiac Fibroblasts are by mechanical tension can up-regulated expression, sST2 conducts
The Decoy receptor of IL-33 blocks the combination of IL-33 and ST2L after being combined with IL-33, weaken what Pathway Activation downstream rose
Cardioprotection, and then Myocardial Remodeling and cardiac dysfunction are aggravated, and with increasing heart failure, myocardial infarction, cardiovascular death
Wait the generation of adverse cardiac events.
At present, the method for detecting ST2 mainly has enzyme-linked immunosorbent assay, immunochromatographic method and chemiluminescence immune assay
Method.Enzyme-linked immunosorbent assay uses horseradish peroxidase or alkali phosphatase enzyme mark antibody, and is catalyzed substrate and generates face
Color change has the characteristics of easy to operate, the stable reagent phase is long, but the detection sensitivity of enzyme-linked immunosorbent assay is relatively low,
It is mainly used in communicable disease screening etc. at present and requires detection sensitivity relatively low project；Immunochromatographic method has operation
Simply, the advantages that detection speed is fast, but it is low there is also sensitivity, and reagent is unstable, less reproducible, it is difficult to carry out quantitative
Shortcoming.Chemiluminescence immunoassay has many advantages, such as easy to operate, and detection speed is fast, high-throughput detection, but there is also it is non-
Phase reaction, detection time is long, criticizes the shortcomings that interior batch variation is big.
To solve the above problems, if the antibody of ST2 can be utilized, using bimolecular fluorescence complementary technology as platform, develop
A kind of ST2 quick detection reagents diagnosed for thrombotic diseases with thromboembolism treatment effect monitoring.Make itself and existing detection reagent
It compares, has many advantages, such as that easy to operate, high sensitivity, free of cleaning, precision is high；It is applied to thrombotic diseases and thrombolysis
The monitoring of therapeutic effect can improve the accuracy rate of thrombotic diseases diagnosis and thromboembolism treatment effect monitoring, then can be by market
Will be widely welcomed and with great market value.
Invention content
For the technical problems in the prior art, the present invention provides a kind of detection examination that can be used for quantitatively detection ST2
Agent box, its method of preparation and use.
The invention is realized by the following technical scheme：
It is a kind of to prepare the method based on bimolecular fluorescence complementary technology ST2 detection kits, include the following steps：
1) anti-ST2 antibody couplings fluorescin N-terminal segment；
2) anti-ST2 antibody couplings fluorescin C-terminal segment；
In the above-mentioned technical solutions, the anti-ST2 antibody is the monoclonal antibody or Anti-TNF-α for ST2 different epitopes
Body.
In said program, the step of the anti-ST2 antibody couplings fluorescin N-terminal segment in, fluorescin N-terminal segment
Mass ratio with anti-ST2 antibody is 1: 1-10.
In said program, the step of the anti-ST2 antibody couplings fluorescin C-terminal segment in, fluorescin C-terminal segment
Mass ratio with anti-ST2 antibody is 1: 1-10.
The prepared ST2 detection reagents based on bimolecular fluorescence complementary technology according to any of the above technical solution
Box.Its mainly form including：
1) the fluorescin N-terminal segment of anti-ST2 antibody couplings；
2) the fluorescin C-terminal segment of anti-ST2 antibody couplings.
The application method of kit described in any of the above technical solution, it is characterised in that：Include the following steps：
1) sample, the fluorescin N-terminal segment of anti-ST2 antibody couplings and anti-ST2 is added in the reacting hole of kit to resist
The fluorescin C-terminal segment of body coupling, hybrid reaction 5-60 minutes；
2) exciting light irradiation reacting hole measures each reacting hole luminous quantity and obtains fluorescence signal value.
The reacting hole of this kit can be microwell plate, micro-fluidic reagent disc, reaction cup, reaction tube etc..
Description of the drawings
Fig. 1 is the ST2 detection kits principle signal provided in an embodiment of the present invention based on bimolecular fluorescence complementary technology
Figure；Reference sign：The anti-ST2 antibody of 1-, 2- bridging agents, 3- fluorescin N-terminal segments, 4- determinands (ST2), the anti-ST2 of 5-
Antibody, 6- fluorescin C-terminal segments, 7- bridging agents.
Fig. 2 is that the ST2 detection kits detection provided in an embodiment of the present invention based on bimolecular fluorescence complementary technology is linear
Areal map.
Fig. 3 is that the ST2 detection kits result provided in an embodiment of the present invention based on bimolecular fluorescence complementary technology is related
Property compares.
Specific embodiment
Below with reference to attached drawing to the present invention the ST2 detection kits based on bimolecular fluorescence complementary technology, prepare and its
Application method is described in detail.
Embodiment 1
Anti- ST2 antibody couplings fluorescin N-terminal segment, with the segment YFPN of yellow fluorescence protein (YFP) 1-154 amino acid
For, specific implementation process is：
1) 0.1mg YFPN albumen is added in centrifuge tube, is matched with 0.05mol/L pH9.5 carbonate buffer solutions (CB)
YFPN albumen is diluted to 1mg/mL by system.
2) final concentration of 1.25% glutaraldehyde is added in draught cupboard.
3) 37 DEG C of water-bath 2h.
4) it is dialysed with desalting column Sephadex G-25 or 0.05mol/LpH9.5 CB and removes excessive glutaraldehyde.
5) the anti-ST2 antibody of 0.1mg is taken, 1mg/mL antibody is prepared with 0.05mol/L pH9.5 CB, by the YFPN eggs of activation
The mixing of white and antibody.
6) 4 DEG C, reaction is overnight.
7) it closes：50 μ L 0.2mol/L lysine solutions are added in, room temperature closing 2h to close remaining aldehyde radical, is terminated anti-
It should.
8) 4 DEG C of placement 2h.
9) polymer insoluble matter is removed by reactant by SephadexG-200 gel columns or with 0.45 μm of filter membrane, used
0.01mol/L pH7.2 PBS dialysis purification conjugates, are diluted to required concentration before use.
Embodiment 2
Anti- ST2 antibody couplings fluorescin C-terminal segment, with the segment of yellow fluorescence protein (YFP) 155-238 amino acid
For YFPC, specific implementation process is：
1) 0.1mg YFPC albumen is added in centrifuge tube, is matched with 0.05mol/L pH9.5 carbonate buffer solutions (CB)
YFPC albumen is diluted to 1mg/mL by system.
2) final concentration of 1.25% glutaraldehyde is added in draught cupboard.
3) 37 DEG C of water-bath 2h.
4) it is dialysed with desalting column Sephadex G-25 or 0.05mol/LpH9.5 CB and removes excessive glutaraldehyde.
5) the anti-ST2 antibody of 0.1mg is taken, 1mg/mL antibody is prepared with 0.05mol/L pH9.5 CB, by the YFPC eggs of activation
The mixing of white and antibody.
6) 4 DEG C, reaction is overnight.
7) it closes：50 μ L 0.2mol/L lysine solutions are added in, room temperature closing 2h to close remaining aldehyde radical, is terminated anti-
It should.
8) 4 DEG C of placement 2h.
9) polymer insoluble matter is removed by reactant by SephadexG-200 gel columns or with 0.45 μm of filter membrane, used
0.01mol/L pH7.2 PBS dialysis purification conjugates, are diluted to required concentration before use.
Embodiment 3
Kit mainly forms：
1) the fluorescin N-terminal segment of anti-ST2 antibody couplings；
2) the fluorescin C-terminal segment of anti-ST2 antibody couplings.
Embodiment 4
Kit application method, includes the following steps：
1) sample, the fluorescin N-terminal segment of anti-ST2 antibody couplings and anti-ST2 is added in the reacting hole of kit to resist
The fluorescin C-terminal segment of body coupling, hybrid reaction 5-60 minutes；
2) exciting light irradiation reacting hole measures each reacting hole luminous quantity and obtains fluorescence signal value.
The reacting hole of this kit can be microwell plate, micro-fluidic reagent disc, reaction cup, reaction tube etc..
Embodiment 5
Kit method evaluation of the present invention：
It is 1. linear
Compound concentration is the ST2 standard solutions of 0ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, 200ng/mL.
20 μ L standard items, the fluorescin N-terminal segment for adding in the anti-ST2 antibody couplings of 50 μ L are separately added into reacting hole, 50 μ L is added in and resists
The fluorescin C-terminal segment of ST2 antibody couplings, 37 DEG C incubate 10 minutes.After incubation, exciting light irradiation reacting hole measures each
Reacting hole luminous quantity obtains fluorescence signal value.
Using fluorescence signal value as ordinate, standard concentration is abscissa, draws standard working curve (see attached drawing 2).
2. accuracy
Recovery test：It is added in the serum specimen of normal person with known quantity ST2 standard items, measures concentration value after adding in
It is compared with the theoretical value of addition, calculates the rate of recovery of ST2.Testing result is as follows：
| Sample number | Add in ST2 concentration (ng/mL) | Measure the concentration (ng/mL) of ST2 | The rate of recovery (%) |
| 1 | 20 | 21.6 | 95.9 |
| 2 | 60 | 58.2 | 102.1 |
| 3 | 120 | 123.5 | 100.6 |
| 4 | 180 | 178.9 | 99.6 |
3. precision
Choose the sample of 3 parts of various concentrations, respectively duplicate measurements 20 times according to the method described in the present invention.According to 20 times
Measurement result calculates average deviation CV values.
4. sensitivity for analysis
The definition of sensitivity for analysis is：Refer to the amount that can be distinguished statistically with zero-dose.It is repeated 20 times measurement
Zero-dose point, calculates its average value (X) and standard deviation (SD), and the concentration value with the calculating of X+2SD is the analysis of the kit
Sensitivity.The sensitivity for analysis of kit of the present invention is 5.0ng/mL.
5. anti-interference
The immunological assay reagents based on bimolecular fluorescence complementary technology of the present invention are detected in interference substance (haemolysis, height
Blood fat, high bilirubin) in the presence of detect sample accuracy.Hemoglobin solutions are taken respectively and are added to ST2 in right amount
In positive serum sample, the content for making hemoglobin in serum is respectively 0.5mg/mL, 1.0mg/mL.By triglycerides solution
Take and be added in ST2 positive serum samples in right amount respectively, the content for making Triglycerides in Serum be respectively 0.5mg/mL,
1.0mg/mL.Bilirubin solution is taken respectively and is added in ST2 positive serum samples in right amount, makes the content of serum mesobilirubin
Respectively 25 μ g/mL, 50 μ g/mL.The resistive samples of ST2 to adding hemoglobin, triglycerides and bilirubin are surveyed
It is fixed.Using the ratio of theoretical concentration and measured concentration as the rate of recovery, the rate of recovery is between 99.7%-104.0%.Show to be based on
The ST2 reagents of bimolecular fluorescence complementary technology are not done when detecting serum sample by hemoglobin, triglycerides, bilirubin
It disturbs.
6. correlation
As shown in figure 3, it is with the correlation of Critical Diagnostics ST2 kits：Y=0.986x-0.121,
R2=0.998.
The present invention is compared with existing method and product, and with detection sensitivity height, specificity is good, cost is relatively low, to detection
The advantages of instrument requirements are low.
The above description is merely a specific embodiment, but protection scope of the present invention is not limited thereto, any
Those familiar with the art disclosed herein technical scope in, the change or replacement that can readily occur in, all
It is covered by the protection scope of the present invention.
Claims (7)
1. a kind of ST2 detection kits, method of preparation and use based on bimolecular fluorescence complementary technology, it is characterised in that：
1) kit mainly forms：The fluorescin C of the fluorescin N-terminal segment of anti-ST2 antibody couplings and anti-ST2 antibody couplings
End fragment；
2) application method：Sample, the fluorescin N-terminal segment of anti-ST2 antibody couplings and anti-are added in the reacting hole of kit
The fluorescin C-terminal segment of ST2 antibody couplings, hybrid reaction 5-60 minutes；
3) detection method：Exciting light irradiates reacting hole, measures each reacting hole luminous quantity and obtains fluorescence signal value.
2. a kind of preparation method of the ST2 detection kits based on bimolecular fluorescence complementary technology, which is characterized in that including as follows
Step：
1) preparation of anti-ST2 antibody couplings fluorescin N-terminal segment；
2) preparation of anti-ST2 antibody couplings fluorescin C-terminal segment.
3. anti-ST2 antibody according to claim 1 is the monoclonal antibody or polyclonal antibody for ST2 different epitopes.
4. fluorescin according to claim 1, including but not limited to green fluorescent protein, blue fluorescent protein, cyan
Fluorescin, yellow fluorescence protein, red fluorescent protein.
5. method prepared by a kind of ST2 detection kits based on bimolecular fluorescence complementary technology according to claim 2,
It is characterized in that, in the anti-ST2 antibody couplings fluorescin N-terminal segment step, fluorescin N-terminal segment and ST2 antibody
Mass ratio be 1: 1-10.
6. method prepared by the ST2 detection kits according to claim 2 based on bimolecular fluorescence complementary technology, special
Sign is, in the anti-ST2 antibody couplings fluorescin C-terminal segment step, fluorescin C-terminal segment and the matter of ST2 antibody
Amount is than being 1: 1-10.
7. reacting hole according to claim 1, including but not limited to microwell plate, micro-fluidic reagent disc, reaction cup, reaction
Pipe.
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| CN201711271870.2A CN108267594A (en) | 2017-11-27 | 2017-11-27 | A kind of ST2 detection kits, method of preparation and use based on bimolecular fluorescence complementary technology |
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| CN201711271870.2A CN108267594A (en) | 2017-11-27 | 2017-11-27 | A kind of ST2 detection kits, method of preparation and use based on bimolecular fluorescence complementary technology |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110208549A (en) * | 2019-07-01 | 2019-09-06 | 北京利德曼生化股份有限公司 | A kind of Soluble growth stimulation 2 albumen sST2 luminescence reagent box of expressing gene |
| WO2021164109A1 (en) * | 2020-02-21 | 2021-08-26 | 苏州旭光科星抗体生物科技有限公司 | Enzyme-linked immunoassay kit for detecting content of soluble st2, and use thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105259353A (en) * | 2015-10-15 | 2016-01-20 | 北京市心肺血管疾病研究所 | Kit and method for detecting sST2 (soluble ST2) in blood of abdominal aortic aneurysm and/or aortic dissection patient |
-
2017
- 2017-11-27 CN CN201711271870.2A patent/CN108267594A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105259353A (en) * | 2015-10-15 | 2016-01-20 | 北京市心肺血管疾病研究所 | Kit and method for detecting sST2 (soluble ST2) in blood of abdominal aortic aneurysm and/or aortic dissection patient |
Non-Patent Citations (2)
| Title |
|---|
| CLIFF I. STAINS ET AL.: "A General Approach for Receptor and Antibody-Targeted Detection of Native Proteins utilizing Split-Luciferase Reassembly", 《ACS CHEMICAL BIOLOGY》 * |
| 黄欣媛等: "蛋白片段互补分析技术研究进展", 《中国生物工程杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110208549A (en) * | 2019-07-01 | 2019-09-06 | 北京利德曼生化股份有限公司 | A kind of Soluble growth stimulation 2 albumen sST2 luminescence reagent box of expressing gene |
| WO2021164109A1 (en) * | 2020-02-21 | 2021-08-26 | 苏州旭光科星抗体生物科技有限公司 | Enzyme-linked immunoassay kit for detecting content of soluble st2, and use thereof |
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Application publication date: 20180710 |